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WSEAS TRANSACTIONS on BIOLOGY
Satish G. Pingale, Madhukar A. Badgujar,
Kiran V. Mangaonkar, Nikos E. Mastorakis
Determination of amoxicillin in human plasma by LC-MS/MS and its application to a bioequivalence study
SATISH G. PINGALE1, MADHUKARA. BADGUJAR1,
KIRAN V. MANGAONKAR1, NIKOS E. MASTORAKIS2
1 Analytical Chemistry Research Laboratory, Mithibai College
Vile Parle (W), Mumbai 400056
2 Military Institutes of University Education, Hellenic Naval Academy
E-mail address: [email protected]Abstract: - An analytical method based on solid phase extraction has been developed and validated for analysis of amoxicillin in human plasma using gemifloxacin as an internal standard. A COSMOSIL 5C18-PAQ column provided chromatographic separation of analytes followed by detection with mass spectrometry. The method involves simple isocratic chromatographic conditions and mass spectrometric detection in the positive ionization mode using an API-3000 system. The proposed method has been validated with linear range of 100–15000 ng/mL for amoxicillin. The intra-run and inter-run precision values are within 3.53% and 5.63% respectively for amoxicillin at the LOQ level. Total elution time was as low as 2 min. This validated method was used successfully for analysis of plasma samples from a bioequivalence study. Key-Words: - Amoxicillin, Antibiotic,Automated solid phase extraction, Tandem mass spectrometry, Human plasma, Bioequivalence study.
1 Introduction
reaches Cmax (8mg/mL) about 2 hours after
administration, exhibits low binding with plasma
proteins (17%), is quickly distributed through the
Amoxicillin is a semi synthetic antibiotic. It is an
body, and has an elimination half-life of 1 hour [3].
analog of ampicillin, with a broad spectrum of
bactericidal activity against many gram-positive
commonly used antibiotic. To understand the
and gram-negative microorganisms. IUPAC name
pharmacokinetic behavior of Amoxicillin in
of the amoxicillin is (2S,5R,6R)-6-[(R)-(-)-2-
humans, a reliable quantitative method is needed.
Several bioanalytical methods are reported to
determine amoxicillin in body fluid by HPLC with
2-carboxylic acid trihydrate. The presence of a
UV [4-10] and fluorescence [11] detection.
benzyl ring in the side chain extends the
Sensitive and selective methods based on
antibacterial activity to gram-negative bacteria [1].
The action mechanism of these antibiotics has not
developed for determination of amoxicillin in
been unequivocally established, but it is thought
bovine milk [12], pig tissues [13] and human
they may interfere with peptidoglycan bacterial cell
wall synthesis in the effected organisms [2].
Different methods of sample preparation
Amoxicillin shows high absorption after
have been applied prior to the chromatographic
oral administration, and this is not altered by the
analysis, mostly based on protein precipitation
concomitant ingestion with food. Amoxicillin
[4,16], liquid-liquid extraction [8,13,14], solid-
E-ISSN: 2224-2902
Issue 1, Volume 9, January 2012
WSEAS TRANSACTIONS on BIOLOGY
Satish G. Pingale, Madhukar A. Badgujar,
Kiran V. Mangaonkar, Nikos E. Mastorakis
techniques like ultra filtration [5,9].
orthophosphoric acid and formic acid were
The method reported by Khuroo et al.
procured from Merck (Mumbai, India). Drug free
describes solid phase extraction of amoxicillin in
(blank) human plasma containing K3-EDTA was
human plasma using LC-MS/MS. The lowest
obtained in-house by enrolling healthy volunteers
LOQ's reported by Khuroo et al., was 170 ng/mL
and taking their consent before bleeding. The
and the linear dynamic range was 170-17000
plasma thus obtained was stored at –20 °C prior to
ng/mL [15]. As per guidelines LOQ should be five-
use. Oasis HLB 30 mg/1 cc solid phase extraction
fold (32 times) below the Cmax value, but
(SPE) cartridges were procured from waters
unfortunately the method reported by Khuroo et al.
(Milford, Mass, USA).
also failed to quantify LOQ of amoxicillin in
human plasma which were observed in the present
bioequivalence study.
2.2 Calibration curve and quality control
The sensitive and selective LC–MS/MS
method was reported by Wen et al. involved
Two separate stock solutions of amoxicillin were
solvent precipitation procedure with methanol.
prepared for bulk spiking of calibration curve and
Separation was achieved on a Lichrospher C18
quality control samples for the method validation
column (150 mm x 4.6 mm ID, 5 µm) using
exercise as well as the subject sample analysis. The
methanol (containing 0.2% of formic acid) and
stock solutions of amoxicillin and gemifloxacin
water (containing 0.2% of formic acid) as a mobile
were prepared in methanol at free base
phase by gradient elution at a flow rate of 1.0
concentration of 1000 µg/mL. Primary dilutions
mL/min. Linear dynamic range was 5-20000
and working standard solutions were prepared from
ng/mL [16]. This method involved precipitation
stock solutions by dilution with water:methanol
method with methanol which caused deposition of
(40:60, v/v). These working standard solutions
matrix on curtain plate of the mass spectrometer
were used to prepare the calibration curve and
and needed cleaning of curtain plate frequently.
quality control samples. Blank human plasma was
Hence, it is necessary to develop and
screened prior to spiking to ensure it was free of
validate a clean, rapid, selective and sensitive
endogenous interference at retention times of
method which can be successfully applied to a
amoxicillin and internal standard gemifloxacin. A
bioequivalence study.
nine point standard curve and four quality control
In the present paper we would like to
samples were prepared by spiking the blank plasma
present a simple solid phase extraction method for
with an appropriate amount of amoxicillin.
Calibration samples were made at concentrations of
gemifloxacin as an internal standard using LC-
100, 200, 750, 1500, 3000, 6000, 9000, 12000 and
MS/MS. The application of this validated method
15000 ng/mL and quality control samples were
in analyzing samples from a bioequivalence study
made at concentrations of 300, 3800, 7600 and
involving amoxicillin is also presented. The
12900 ng/mL for amoxicillin.
greatest advantage of this method over all those
referenced is the simple and clean SPE procedure
with short runtime. Linear dynamic range for
2.3 Sample preparation
amoxicillin was sufficient for application in
A 0.5 mL aliquot of human plasma sample was
bioavailability-bioequivalence studies.
mixed with 20 µL of internal standard working
solution (35 µg/mL of gemifloxacin). To this 0.5
mL of 20% orthophosphoric acid was mixed.
2 Experimental
Sample mixture was loaded onto Oasis 30 mg/1 cc
extraction cartridge pre-conditioned with 1 mL
methanol followed by 1 mL water. The extraction
2.1 Chemicals and reagents
cartridge was washed with 1 mL of methyl tertiary
The reference standards of amoxicillin and
butyl ether. Amoxicillin and gemifloxacin were
eluted with 1 mL of the mobile phase. 5 µL of the
Laboratories (Mumbai, India). High purity water
eluant was injected into the LC–MS/MS system
was prepared in-house using a Milli-Q A10
through the autosampler.
gradient water purification system (Millipore,
Bangalore, India). LC grade methanol and methyl
tertiary butyl ether were purchased from J.T. Baker
E-ISSN: 2224-2902
Issue 1, Volume 9, January 2012
WSEAS TRANSACTIONS on BIOLOGY
Satish G. Pingale, Madhukar A. Badgujar,
Kiran V. Mangaonkar, Nikos E. Mastorakis
2.4 Liquid chromatography and mass
for selectivity, sensitivity, linearity, precision and
spectrometric conditions
accuracy, recovery, dilution integrity, partial
2.4.1 Chromatography
volume, matrix effect, reinjection reproducibility
Chromatographic separation was carried out on a
and stability. Selectivity was performed by
Shimadzu HPLC system (Kyoto, Japan) using a
analyzing the human blank plasma samples from
COSMOSIL 5C18-PAQ C
six different sources (or donors) with two more
18 column (50×4.6 mm
i.d., 5 µm) purchased from Chromatopak (Thermo
lots, each of haemolyzed and lipemic to test for
interference at the retention time of amoxicillin and
conditions. The mobile phase was 0.2% formic acid
gemifloxacin. The intrarun (within a day) and
in water–0.2% formic acid in methanol (20:80, v/v)
interrun (on three different days) accuracy was
and set at a flow rate of 0.6 mL min-1. The column
determined by replicate analysis of quality control
was maintained at room temperature. The HPLC
samples (n = 6) at the LOQ (limit of
eluent was introduced directly into the positive
quantification), LQC (low quality control), MQC
electrospray ionization source and the total run
(medium quality control), M1QC (middle quality
time for each sample analysis was 2 min.
control), HQC (high quality control) and ULOQ
(upper limit of quantification) levels. The %CV
should be less than 15% and accuracy (%RE)
2.4.2 Mass spectrometry
should be within 15% except the LOQ where it
The plasma concentration of amoxicillin was
should be within 20%.
quantified using a Sciex API 3000 triple
Accuracy is defined as the percent relative
error (%RE) and was calculated using the formula
Biosystems, MDS Sciex, Concord, Canada)
%RE = (E - T) (100/T) where E is the
equipped with a Turbo Ion Spray interface to
experimentally determined concentration and T is
generate the positively charged ions. Ion spray
the theoretical concentration. Assay precision was
voltage was 5500 V with a turbo gas temperature at
calculated by using the formula %CV = (SD/M)
450°C. The operating conditions were optimized by
(100) where M is the mean of the experimentally
flow injection of a mixture of all analytes and were
determined concentrations and SD is the standard
as follows: nebulizing gas flow 13 L min-1;
auxiliary gas flow 8 L min-1; collision activated
The extraction efficiencies of amoxicillin
dissociation (CAD) gas was set at 7 psi and curtain
and gemifloxacin were determined by analysis of
gas flow 15 L min-1. Compound-dependent
six replicates at low, medium, middle and high
parameters for amoxicillin and gemifloxacin were:
quality control concentrations for amoxicillin and
orifice voltage 50 eV, ring voltage 100 eV.
at one concentration for the internal standard
Quantitation was performed in multiple reaction
gemifloxacin. The percent recovery was evaluated
monitoring (MRM) mode employing collision
by comparing the peak area of extracted analytes to
energies of 16 eV for amoxicillin and 30 eV for
the peak area of non extracted standards (sample
gemifloxacin with dwell times of 250 ms for ion
spiked in mobile phase).
transitions m/z 366.2→ m/z 348.8 (amoxicillin) and
The dilution integrity experiment was
performed with an aim to validate the dilution test
respectively. Quadrupoles, Q1 and Q3 were set to
to be carried out on higher analyte concentrations
unit resolution. Automated data acquisition and
above upper limit of quantification (ULOQ), which
analysis were performed using the Analyst software
may be encountered during real subject samples
(version 1.4.2).
analysis. Dilution integrity experiment was carried
For quantification peak area ratios of the
out at 1.7 times the ULOQ concentration. Six
target ions those of the internal standard were
replicates each of 1/2 and 1/4 concentrations were
compared with weighted (1/x2) least squares
prepared and their concentrations were calculated
calibration curves in which the peak area ratios of
by applying the dilution factor 2 and 4 against the
the calibration standards were plotted versus their
freshly prepared calibration curve.
The partial volume experiment was
performed to validate the method, for application in
case of insufficient volume of plasma in real
2.5 Validation
subject sample. Partial volume experiment was
A through and complete method validation of
performed on middle quality control (M1QC)
amoxicillin in human plasma was done following
concentration level. Six replicates each of half and
USFDA guidelines [17]. The method was validated
quarter volume of total volume of plasma required
E-ISSN: 2224-2902
Issue 1, Volume 9, January 2012
WSEAS TRANSACTIONS on BIOLOGY
Satish G. Pingale, Madhukar A. Badgujar,
Kiran V. Mangaonkar, Nikos E. Mastorakis
Application
concentrations were calculated by applying the
bioequivalence study
dilution factor 2 and 4 against the freshly prepared
The validated method has been successfully applied
calibration curve.
to the analysis of amoxicillin concentrations in
To study the effect of matrix on analyte
twenty four healthy, adult, human male volunteers
quantification with respect to consistency in signal
which were enrolled in a bioequivalence study. One
suppression, matrix effect was checked with six
amoxicillin tablet (875 mg) was orally administered
different lots of plasma. Two replicates each of
to volunteers rang ranging in age from 18 to 45
LQC and HQC were prepared from six different
years as per the inclusion criteria for the study
lots of plasma (total 24 QC samples) and checked
under fasting conditions. Volunteers who have used
for %CV which should be less than 10% at LQC
any prescribed medication during last 2 weeks or
over the counter medicinal products during the last
reproducibility,
week preceding the first dose were excluded from
initially LQC and HQC samples were injected and
the study. Seven days of wash-out period were kept
after analysis of these samples system hardware
between the two periods of the study.
was deactivated for two hours, after three hours
The bioequivalence study was based on a
again system hardware was activated and
balanced, open label, analyst blind, single centre,
equilibrated. Then same samples were reinjected.
randomized, crossover, two treatment, two periods,
Original values were compared with reinjected
two sequences and single dose design. A test
values with respect to %Change, which should be
formulation containing Amoxicillin 875 mg with
the reference formulation Augmentin® was
As a part of the method validation, stability
evaluated. The study was conducted according to
was evaluated in stock solutions and in plasma
current GCP guidelines after signed consent of the
under different conditions, simulating the same
volunteers following approved by an authorized
conditions, which occurred during study sample
Ethics committee. In total, 20 time points after
handling and analysis. Stock solution stability was
blood collection per period were evaluated post-
performed by comparing area response of stability
dosing including the pre-dose sample. The blood
sample of analyte and internal standard with the
samples were collected in separate vacutainers
area response of sample prepared from fresh stock
containing K3-EDTA. The plasma from these
solutions. Stability studies in plasma were
samples was analyzed by LC-MS-MS.
performed at LQC and HQC concentration level
using six replicates at each level. Analyte was
computed using the WinNonlin Software version
considered stable if the %Change is less than 15%
5.2 (Pharsight Corporation, California, USA) and
as per USFDA guidelines. Analyte was tested using
90% confidence interval was computed using SAS
the quality control samples whenever appropriate.
Software version 9.2 (SAS Institute, Cary, USA).
The stability of spiked human plasma stored at
room temperature (bench top stability) was
evaluated for 12 h. The stability of spiked human
3 Results and discussion
plasma stored at -70 °C in coolant (coolant
stability) was evaluated for 24 h. The autosampler
sample stability was evaluated by comparing the
3.1 Method development
extracted plasma samples that were injected
During method development different options were
immediately (time 0), with the samples that were
evaluated to optimize detection parameters,
re-injected after keeping in the auto sampler at 10
chromatography and sample extraction.
°C for 24 h. The freeze–thaw stability was
conducted by comparing the stability samples that
had been frozen at –20 °C and thawed three times,
3.1.1 Mass spectra
with freshly spiked quality control samples. Six
The LC–MS/MS were tested to obtain sensitivity
aliquots of each low and high concentration were
and signal stability during infusion of the analyte in
used for the freeze–thaw stability evaluation. For
the continuous flow of mobile phase to electrospray
long term stability evaluation the concentrations
ion source operated both polarities at a flow rate of
obtained after 15, 30, 45, 60 and 90 days intervals
10 µL/min. The amoxicillin gave better results in
were compared with initial concentrations.
positive ion mode. The predominant peaks in the
E-ISSN: 2224-2902
Issue 1, Volume 9, January 2012
WSEAS TRANSACTIONS on BIOLOGY
Satish G. Pingale, Madhukar A. Badgujar,
Kiran V. Mangaonkar, Nikos E. Mastorakis
gemifloxacin corresponds to the MH+ ions at m/z
amoxicillin and gemifloxacin scanned in
366.2 and 390.0 respectively. Product ions of
Fig.1 Parent ion mass spectrum of amoxicillin Fig.2 Product ion mass spectrum of amoxicillin.
E-ISSN: 2224-2902
Issue 1, Volume 9, January 2012
WSEAS TRANSACTIONS on BIOLOGY
Satish G. Pingale, Madhukar A. Badgujar,
Kiran V. Mangaonkar, Nikos E. Mastorakis
Fig.3 Parent ion mass spectrum of gemifloxacin

Fig.4 Product ion mass spectrum of gemifloxacin
quadrupole 3 after a collision with nitrogen in
The mobile phase containing 2mM ammonium
quadrupole 2 had an m/z of 348.8 and 371.9
acetate:acetonitrile
ammonium acetate:methanol (20:80v/v) exhibited
better separation, but response was very low, which
was insufficient to quantify LOQ. We had tried
3.1.2 Chromatography
mobile phase 0.2% formic acid in water solution in
Initially, a mobile phase containing acetic acid
combination with 0.2% formic acid in methanol
solution and acetonitrile in varying combinations
and 0.2% formic acid in acetonitrile in varying
was tried in which poor peak shape was observed.
combinations. Use of 2mM ammonium acetate
E-ISSN: 2224-2902
Issue 1, Volume 9, January 2012
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Satish G. Pingale, Madhukar A. Badgujar,
Kiran V. Mangaonkar, Nikos E. Mastorakis
with 0.2% formic acid in water in combination with
In the state of nonionic forms, the strong binding of
methanol improves peak shape but response was
analytes to the copolymer of the SPE cartridge
low. Finally we received the best signal for
enabled sufficient clean-up and suitable eluting
amoxicillin and gemifloxacin using a mobile phase
solution helped to elute analytes with more
containing 0.2% formic acid in water solution in
efficiency. So the method was optimized to achieve
combination with 0.2% formic acid in methanol
maximum extraction efficiency. The amoxicillin is
(20:80 v/v) as there was almost a two fold increase
approximately 17% protein-bound, so prior to
in its area count as compared to the mobile phase
extraction it is necessary to release the amoxicillin
containing 0.2% formic acid in water solution in
from protein binding. So orthophosphoric acid was
combination with acetonitrile. Moreover, a marked
added in the sample before extraction. for breaking
improvement in the peak shape of amoxicillin and
protein binding. Different types of cartridges such
gemifloxacin was also observed using this mobile
as Phenomenex (Strata-X, Strata-X-C, Strata-
phase combination.
XCW, Strata-XAW) and Oasis HLB were tried.
Short length columns such as, COSMOSIL
Finally we choose an Oasis HLB 30 mg/1 cc
5C18-PAQ (50 mm x 4.6 mm, 5 µm), Symmetry
cartridge. It was difficult to find a compound which
Shield RP18 (50 mm x 2.1 mm, 3.5 µm), Inertsil
could ideally mirror the analytes to serve as a good
C18 (50 mm x 4.6 mm, 5 µm), HyPURITY C18
IS. Several compounds were investigated to find a
(50 mm x 4.6 mm, 5 µm) and HyPURITY Advance
suitable IS, and finally gemifloxacin of the same
(50 mm x 4.0 mm, 5 µm) were tried during the
class of compound was found most appropriate for
method development. In Symmetry Shield RP18
the present purpose. There was no significant effect
columns poor chromatography was observed.
of IS on analyte recovery, sensitivity or ion
Inertsil C18, HyPURITY Advance and HyPURITY
suppression. The results of method validation using
C18 columns gave a relatively good peak shape but
gemifloxacin as the IS were acceptable in this study
the response was low. The best signal was obtained
based on FDA guidelines. The high recovery and
using the COSMOSIL 5C18-PAQ (50 mm x 4.6
selectivity was observed in the solid phase
mm, 5 µm) column. It gave satisfactory peak
extraction method which was used in the present
shapes for all the analytes and a flow rate of 0.6
mL/min reduced the run time to 2.0 min.
These optimized detection parameters,
Introducing such a high flow directly in to the
ionization source affects evaporation of solvents
procedure resulted in reduced analysis time with
which further causes improper ionization and
accurate and precise detection of amoxicillin in
reduces response, hence splitter was utilized to
control direct flow in the ionization source.
3.1.3 Extraction 3.2 Method validation
Prior to LC injection, the co-extracted proteins
should be removed from the prepared solution.
Several organic solvents were employed to extract
3.2.1 Selectivity and sensitivity
analytes from the plasma sample. In the tested
Representative chromatograms obtained from blank
solvents (ethyl acetate, chloroform, hexane,
plasma, plasma spiked with internal standard and
dichloromethane, methyl tertiary butyl ether and
plasma spiked with LOQ standard for amoxicillin
acetonitrile) samples were not clean and poor
and gemifloxacin is presented in Fig. 5.
chromatography observed.
E-ISSN: 2224-2902
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WSEAS TRANSACTIONS on BIOLOGY
Satish G. Pingale, Madhukar A. Badgujar,
Kiran V. Mangaonkar, Nikos E. Mastorakis
Fig.5 Representative chromatograms of amoxicillin (left) and gemifloxacin (right) in human plasma. (A) Blank plasma (B) Plasma spiked with internal standard and (C) LLOQ

The mean %interference observed at the retention
the present method. All the values obtained below
time of analytes between eight different lots of
100 ng/mL for amoxicillin were excluded from
human plasma including haemolyzed and lipemic
statistical analysis as they were below the LOQ
plasma containing K3-EDTA as the anti-coagulant
values validated for amoxicillin.
calculated was 0.00% for amoxicillin and
gemifloxacin, which was within acceptance
criteria. Six replicates of extracted samples at the
Linearity, precision accuracy,
LOQ level in one of the plasma samples having
recovery
least interference at the retention time of
The peak area ratios of calibration standards were
amoxicillin was prepared and analyzed. The %CV
proportional to the concentration of amoxicillin in
of the area ratios of these six replicates of samples
each assay over the nominal concentration range of
was 6.68% for amoxicillin confirming that
100–15000 ng/mL. The calibration curves appeared
interference does not affect the quantification at
linear and were well described by least-squares
LOQ level. Utilization of selected product ions for
linear regression lines. As compared to the 1/x
each compound enhanced mass spectrometric
weighing factor, a weighing factor of 1/x2 properly
selectivity. The product ions of m/z 366.2 and
achieved the homogeneity of variance and was
390.0 were hence concluded to be specific for
chosen to achieve homogeneity of variance. The
amoxicillin and gemifloxacin.
correlation coefficient was ≥0.9900 for amoxicillin.
The LOQ for amoxicillin was 100 ng/mL.
The observed mean back-calculated concentration
The intra-run precision and intra-run accuracy
with accuracy (%RE) and precision (%CV) of four
(%RE) of the LOQ plasma samples containing
linearities analyzed during method validation are
amoxicillin was 3.53 and -1.81, respectively. We
given in table 1. The deviation of the back
obtained the mean Cmax of amoxicillin, for test and
calculated values from the nominal standard
reference formulations were 8521.98 and 8696.75
concentrations were less than 15% except LOQ
ng/mL, respectively. As per guidelines LOQ should
where it should be less than 20%. This validated
be below 32 times (five-fold) of Cmax. So for
linearity range justify the concentration observed
amoxicillin, the LOQ was easily, quantified using
during real sample analysis.
E-ISSN: 2224-2902
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Satish G. Pingale, Madhukar A. Badgujar,
Kiran V. Mangaonkar, Nikos E. Mastorakis
Table 1 Summary of calibration curve standards

Nominal concentration
Mean back calculated
concentration (ng/mL)
CV: coefficient of variation; RE: relative error.
individual assay results of replicate (n = 6) quality
The inter-run precision and accuracy were
control of two separate batch runs analyzed on the
determined by pooling all individual assay results
same day. The intra-run precision (%CV) and intra-
of replicate (n = 6) quality control over three
run accuracy (%RE) was ≤3.53% and ≤-3.99%
separate batch runs analyzed on three different
respectively for amoxicillin. All these data
days. The inter-run precision and inter-run
presented in Table 2 indicate that the method is
accuracy (%RE) was ≤5.63% and ≤-8.43%
precise and accurate.
respectively for amoxicillin. The intra-run precision
and accuracy were determined by pooling all
Table 2 Intra-run and inter-run precision and accuracy (n = 6) of amoxicillin in human plasma

Analytes Level
HQC 895241 333162
Gemifloxacin M1QC 749219
A: Area of unextracted sample; B: Area of extracted sample; Mean recovery was found to be 37.25% for amoxicillin and 48.83 for gemifloxacin; CV: coefficient of variation.
and HQC were 4.45% and 2.47% respectively for
Six aqueous replicates (samples spiked in
mobile phase) at low, medium, middle and high
amoxicillin. Hence minor suppression of analyte
quality control concentration levels for amoxicillin
signal due to endogenous matrix interferences did
were prepared for recovery determination and the
not affect the quantification of amoxicillin.
areas obtained were compared versus the areas
Reinjection reproducibility exercise was
obtained for extracted samples ( shown in Table 3)
performed to check weather the instrument
of the same concentration levels from a precision
performance remains unchanged after hardware
and accuracy batch run on the same day. The mean
deactivation due to any instrument failure during
recovery for amoxicillin was 37.25% with a
real subject sample analysis. %Change is less than
precision of 4.85%. The mean recovery for
4.13% for LQC and HQC level concentration;
gemifloxacin was 48.83% with a precision of
hence batch can be reinjected in case of instrument
4.81%. This indicates that the extraction efficiency
failure during real subject sample analysis.
for the amoxicillin as well as gemifloxacin was
Stock solution stability was performed to
consistence and reproducible.
check stability of amoxicillin and gemifloxacin in
stock solutions prepared in water : methanol
(80:20) and stored at 2-8 °C in refrigerator. The
3.2.3 Dilution integrity and partial volume
freshly prepared stock solutions were compared
The mean back calculated concentrations for 1/2
with stock solutions prepared before 30 days. The
and 1/4 dilution samples were within 85-115% of
%Change for amoxicillin and gemifloxacin were
their nominal. The %CV for 1/2 and 1/4 dilution
2.22% and 1.99% respectively indicates that stock
samples were 1.52% and 1.53% respectively. The
solutions were stable at least for 30 days.
mean back calculated concentrations for half and
Bench top, coolant and autosampler
quarter partial volume samples were within 85-
stability for amoxicillin was investigated at LQC
115% of their nominal. The %CV for half and
and HQC levels. The results revealed that
quarter partial volume samples were 2.16% and
amoxicillin was stable in plasma for at least 12 h at
1.31% respectively.
room temperature, 24 h in coolant at -70 °C and 24
h in an autosampler at 10 °C. It was confirmed that
repeated freezing and thawing (three cycles) of
3.2.4 Matrix effect, reinjection reproducibility
plasma samples spiked with amoxicillin at LQC
and stabilities.
and HQC levels did not affect their stability. The
The assessment of matrix effect constitutes an
long term stability results also indicated that
important and integral part of validation for
amoxicillin was stable in matrix for up to 90 days
at a storage temperature of –20 °C. The results
pharmacokinetics studies. The results found were
obtained from all these stability studies are
well within the acceptable limits as the %CV of the
tabulated in Tables 4.
area ratios of post spiked recovery samples at LQC
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Satish G. Pingale, Madhukar A. Badgujar,
Kiran V. Mangaonkar, Nikos E. Mastorakis
Table 4 Stability results for montelukast